CN103649302A - Cell culture device, device for long-term monitoring of cell culture, method for long-term cell culture, and method for long-term monitoring of cell culture - Google Patents
Cell culture device, device for long-term monitoring of cell culture, method for long-term cell culture, and method for long-term monitoring of cell culture Download PDFInfo
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Abstract
Provided are a cell culture device, a device for long-term monitoring of cell culture, a method for long-term cell culture, and a method for long-term monitoring of cell culture, with which it is possible to culture and monitor cells continuously under uniform environmental conditions and to trace a specific cell history (genealogy) without the changes in physical state that accompany aging of culture cells. The cell culture device comprises a cell culture substrate, a semipermeable membrane, and culture liquid feed means, wherein the cell culture substrate has at the surface a thin culture groove for carrier culture of cells and a thick discharge groove for discharge of cells that have been carrier cultured in the culture groove; both ends of the culture groove are connected to the discharge groove; the discharge groove is thicker and deeper than the culture groove; and the semipermeable membrane covers the culture groove and the discharge groove of the cell culture substrate. The culture liquid feed means are capable of continuously feeding culture liquid to the cell culture substrate covered by the semipermeable membrane.
Description
Technical field
The present invention relates to cell culture apparatus, cell cultures long-term observation device, cell long-period culture method and cell cultures long-term observation method.
Background technology
All the time, when cell being put into dish ware etc. and examining under a microscope cell, along with the process of time, cell consumption nutrition and put aside refuse, therefore cause pericellular environment to change, be difficult to go through continuous cultivation, the observation of the cell of several generations.And, in this situation, because being geometricprogression, cell breeds, and therefore exist and be difficult to the such problem of the specific cell of tracing study.
Therefore,, as the method for long-term cultured, for example, the inventor has proposed the method (non-patent literature 1) that cell is put into the container of micron-scale and made with cell treatment technologies such as light tweezers, a part of cell to be removed outside measuring system.But for the method, once the quantity of mobile cell is few, and experimenter need to select and remove cell one by one, and therefore the burden of work is large, in fact the longest 10 cultured continuously of left and right from generation to generation that also can only reach.
On the other hand, recently, as the method for long-term cultivation, observation of cell, pay close attention to the method (non-patent literature 2) of a kind of being called as " Mother machine ".In the method, on substrate, form the little groove (1 μ m is wide, 25 μ m are long) of width of the groove that width is large (100 μ m are wide), palpus shape.And, cell is put into the groove that this width is little, and the nutrient solution that makes to flow in groove that width is large, utilize nutrient solution to wash away along with cell proliferation and be expressed into the cell the groove that width is large from the little groove of width, thereby remove continuously unwanted cells, and can go through 200, with Shangdi, cultivate the cell in the groove that width is little from generation to generation.
[formerly technical literature]
[non-patent literature]
Non-patent literature 1:Wakamoto, Y.et al. (2001) Fres ' J Anal Chem; Wakamoto, Y.et al. (2005) Analyst
Non-patent literature 2:Current Biology, 22June2010, Pages1099-1103 " Robust Growth of Escheruchia coli. " Ping Wang et al.
Summary of the invention
[problem that invention will solve]
But " Mother machine " is designed to, a side's of the groove that the width of palpus shape is little end is blocked, even if removed cultured continuously unwanted cells, in the little groove of this width, residual cell must be also old cell (parent cell).That is, in " Mother machine ", there is not the design that suppresses the variation of the physiological status of generation along with the aging of culturing cell, thereby have the problem of the variation that is inevitably attended by the physiological status producing along with the aging of observed cell.
In addition,, in " Mother Machine ", the replacing of the nutrient solution in the little groove of width that pericellular culture environment causes by the diffusion by from the large groove of width is carried out.In the method, in the situation that the length of the little groove of width is formed longlyer, there is such problem: in the little groove of width, near the region of the large groove of width with away from the envrionment conditions in the region of the large groove of width, larger variation occurs.Therefore, for example, at observation of cell during to the response of medicine etc., because the difference of envrionment conditions causes that the result of the response of culturing cell fluctuates, thereby cause being difficult to realizing check accurately.
The present invention completes in view of situation as above, its object is the cell culture apparatus, cell cultures long-term observation device, cell long-period culture method and the cell cultures long-term observation method that provide following,, the variation of the physiological status of not following the aging of culturing cell and producing, can be under uniform envrionment conditions long-term cultivation, observation of cell continuously, thereby the history (pedigree) that can follow the trail of specific cell.
[for solving the means of problem]
In order to solve above-mentioned problem, cell culture apparatus of the present invention has cell cultures substrate, semi-permeable membranes, the feed mechanism of nutrient solution, described cell culture apparatus is characterised in that, cell cultures substrate has thin cultivation for keeping culturing cell with groove with for by the thick discharge groove that keeps cultured cells to discharge in groove in this cultivation on surface, and, described cultivation is connected with groove with discharge with the two ends of groove, discharge is cultivated and ratio cultivation groove depth thick with groove with groove ratio, semi-permeable membranes is for covering groove and the discharge groove for cultivation of cell cultures substrate, the feed mechanism of nutrient solution can be supplied with nutrient solution continuously to the cell cultures substrate being covered by semi-permeable membranes.
In this cell culture apparatus, be preferably, semi-permeable membranes can be by vitamin H-avidin in conjunction with covering cell cultures substrate.
In this cell culture apparatus, more preferably, the feed mechanism of described nutrient solution has liquor charging liner.
Cell cultures long-term observation device of the present invention is characterised in that, this cell cultures long-term observation device possess described cell culture apparatus and can observation of cell plate on the microscopic examination mechanism of cell.
In this cell cultures long-term observation device, be preferably, microscopic examination mechanism is inverted microscope.
Cell long-period culture method of the present invention is the method for utilizing described cell culture apparatus long-term cultured.In addition, described cell long-period culture method is characterised in that, this cell long-period culture method comprises: the operation in groove for cultivation that the cell of expectation is remained on to cell cultures substrate; The operation of groove is used in the cultivation that utilizes semi-permeable membranes to cover cell cultures substrate with groove and discharge; Utilize feed mechanism to carry continuously nutrient solution to cell cultures substrate, via semi-permeable membranes, to the cultivation that is maintained at cell cultures substrate, with the cell in groove, supply with nutrient solution, and, utilize the discharge that is connected at the two ends with cultivating with groove with nutrient solution mobile in groove to cultivate a part with the cell in groove to discharge the operation with groove discharge.
Cell cultures long-term observation method of the present invention is to utilize the method for described cell cultures long-term observation device long-term cultivation, observation of cell.In addition, described cell cultures long-term observation method is characterised in that, described cell cultures long-term observation method comprises: the operation in groove for cultivation that the cell of expectation is remained on to cell cultures substrate; The operation of groove is used in the cultivation that utilizes semi-permeable membranes to cover cell cultures substrate with groove and discharge; Utilize feed mechanism to carry continuously nutrient solution to cell cultures substrate, via semi-permeable membranes, to the cultivation that is maintained at cell cultures substrate, with the cell in groove, supply with nutrient solution, and, utilize the discharge that is connected at the two ends with cultivating with groove with nutrient solution mobile in groove will cultivate a part with the cell in groove to discharge the operation with groove discharge; Utilize the operation of the cell on microscopic examination mechanism observation of cell plate.
[invention effect]
According to the present invention, can carry out long-term cultured continuously, thereby can not produce the variation of the physiological status producing along with the aging of culturing cell, can cultured continuously cell under uniform envrionment conditions or under the Condition of Environment Changes being controlled, and energy long-term observation growth conditions, can observe to follow the trail of for a long time the mode of the growth of specific cells, the frequency that therefore can carry out the distribution of cell size frequency, growth rate distributes, the frequency of generation time distributes, mensuration, the cytophyletic mensuration of the autocorrelative function of protein expression level.
Accompanying drawing explanation
Fig. 1 (A) is the part enlarged view exemplified with an embodiment of the cell cultures substrate of formation cell culture apparatus of the present invention.Fig. 1 (B) is the A-A ' sectional view of Fig. 1 (A), and Fig. 1 (C) is the B-B ' sectional view of Fig. 1 (A).
Fig. 2 is the part enlarged view exemplified with another embodiment of the cell cultures substrate of formation cell culture apparatus of the present invention.
Fig. 3 (A) is the overall diagram exemplified with an embodiment of cell culture apparatus of the present invention, and a part is represented with section.Fig. 3 (B) is the sketch chart exemplified with the using method of semi-permeable membranes.
The schematic diagram of the state of cell when Fig. 4 supplies with nutrient solution for the cell cultures substrate exemplified with to being covered by semi-permeable membranes, (A) is vertical view, is (B) sectional view.
Fig. 5 is the overall diagram exemplified with another embodiment of cell culture apparatus of the present invention, and a part is represented with section.
Fig. 6 is the overall diagram exemplified with an embodiment of cell cultures long-term observation device of the present invention, and a part is represented with section.
Fig. 7 records a part for the consecutive image of the situation of the cell in groove for the cultivation that is present on cell cultures substrate.
Fig. 8 has 55 variations of cell size of generation that obtain by image analysis, a clone and the figure of the variation of the GFP expression level (IC) of cell interior for drawing.
Fig. 9 observes, measures GFP mean fluorecence briliancy in the cell of all cells in all moment of observing and determines the figure (embodiment 4) of result of the frequency distribution of the GFP expression level of inferring out accordingly for expressing the colibacillary time delay of GFP.
Figure 10 observes the figure (embodiment 5) of the result of the frequency distribution that determines cell size for expressing the colibacillary time delay of GFP.
Figure 11 observes the figure (embodiment 6) of the result of the frequency distribution that determines growth rate for expressing the colibacillary time delay of GFP.
Figure 12 observes the figure (embodiment 7) of the result of the frequency distribution that determines generation time for expressing the colibacillary time delay of GFP.
Figure 13 observes the figure (embodiment 8) of the result of the autocorrelative function that determines protein expression level for expressing the colibacillary time delay of GFP.
Figure 14 observes for expressing the colibacillary time delay of GFP the figure (embodiment 9) that determines the result that the frequency in cell fission age distributes.
Figure 15 observes for expressing the colibacillary time delay of GFP the figure (embodiment 10) that determines the cytophyletic result of observing in groove an observation.
Figure 16 (A) is less than the photo of taking from glass substrate top of the state of the cell in the situation of groove for the cultivation of cell size for the surface that is illustrated in glass substrate is formed with groove depth.Figure 16 (B) has the photo of taking from glass substrate top of the state of the cell in the situation of groove for 2 times of above big or small cultivations of cell size for the surface that is illustrated in glass substrate is formed with groove depth.
Embodiment
Cell culture apparatus of the present invention has the feed mechanism of cell cultures substrate, semi-permeable membranes, nutrient solution.
Fig. 1 (A) is the part enlarged view exemplified with an embodiment of the cell cultures substrate of formation cell culture apparatus of the present invention.Fig. 1 (B) is the A-A ' sectional view of Fig. 1 (A), and Fig. 1 (C) is the B-B ' sectional view of Fig. 1 (A).
Cell cultures substrate is formed with the cancellate groove pattern that thin cultivation is connected with groove M with thick discharge with the two ends of groove L from the teeth outwards.Cultivate with groove L and discharge and intersect with approximate right angle with groove M.
Cultivate with groove L as cell being remained in groove and the region performance function that can carry out continuous cultivation is discharged with groove M as cultivating with being washed away and eject by cultured cells appropriateness the region performance function that the environment in groove L is used in adjusting cultivation in groove L by being held in.
In the present invention, continuous cultivation refers to go through the cell cultures of a plurality of generations, in the present invention, by controlling pericellular environment, can realize the long term cell cultures of going through more than 200 generations.
Cell as object of the present invention can be desired cell, and the kind of cell is circumscribed not.Particularly, for example, comprise the cells such as bacterial cell such as stem cell from people or non-human animal's separate tissue, skin cells, mucomembranous cell, liver cell, islet cells, neurocyte, chondrocyte, endotheliocyte, epithelial cell, osteocyte, myocyte etc., vegetable cell, insect cell, intestinal bacteria, can cultivate one kind or two or more cell wherein.
Cultivation is with groove L owing to being for cell being held in to the groove in groove, and therefore, prerequisite is the size that groove depth L1 and well width L2 are greater than institute's cultured cells.Particularly, cultivate groove depth L1 with groove L and can be designed to than the size of cell large 1 times～2 times, be preferably 1 times～1.5 times.And, cultivate well width L2 with groove L and can be designed to than the size of cell large 1 times～3 times, be preferably 1 times～2 times.
In this said " size of cell ", in the situation that be roughly spherical cell, the diameter (maximum length) of cell of take is benchmark.And, for example, in the situation that be the cell beyond elongated bar-shaped equal sphere, can determine according to the thickness (width) that cell is loaded to the cell under the state on substrate the size of cell, in this case, cultivate groove depth L1 with groove L and can be designed to large 1 times～2 times of thickness (width) than cell, well width L2 can be designed to large 1 times～3 times of thickness (width) than cell.
And the known size that the size of cell is recorded in also can reference literature etc., also can the result by the size of the mensuration cells such as microscope determine according to reality.
When cultivating when the groove depth L1 of groove L and well width L2 are in described scope, can by cytotostatic remain on and cultivate with in groove L, thereby can carry out continuous cultivation, the observation of cell.In addition, when cultivating groove depth L1 with groove L and have than large 2 times of the size of cell big or small, cell may not can rest on to be cultivated with in groove L and flow out with groove M to discharging, and is difficult to cell to be held in cultivation with in groove L and cultivate continuously.And when cultivating well width L2 with groove L and have than large 3 times of the size of cell big or small, semi-permeable membranes is also adhered to be cultivated with in groove, may cause damaging by pressure cell or cell and not go out with concentrated flow to discharge, is difficult to cultivate continuously.
In addition, cultivate with the length of groove L can be according to the size of cell, should in cultivating with groove L, keep suitable designs the such as quantity of cultured cells.Particularly, for example, can illustration the scope of 10 μ m～100 μ m left and right, the preferably scope of 30 μ m～100 μ m left and right.
On the other hand, discharge and form slightlyer, dark than cultivating with groove L with groove M.That is, discharge and to form to such an extent that be greater than groove depth L1 and the well width L2 that cultivates use groove L with the groove depth M1 of groove M and well width M2.At this, as shown in Figure 1, " discharging the groove depth M1 with groove M " refers to from cultivating the height location of the bottom land of using groove L and to discharge, uses the distance of the bottom land of groove M.
Discharge with the groove depth M1 of groove M and well width M2 can according to cultivate with groove L keep the size, quantity of cultured cells, in the speed of the moving cell culture fluid in cell cultures substrate upper reaches, can wash away cultivate by the scope of the cell in groove L in suitably design.As benchmark roughly, discharge the size of 3 times～50 times that is preferably the size of cell with the groove depth M1 of groove M.And, with regard to discharge with groove M with cultivate with regard to the relation of groove L, can illustration discharging with the groove depth M1 of groove M be the degree of depth of 1.5 times～50 times of cultivating the groove depth L1 that uses groove L.Particularly, discharge with the groove depth M1 of groove M and can the scope about 5 μ m～50 μ m be shown preference.
In addition, discharge with the well width M2 of groove M and have and groove depth M1 equal extent or its above size.With regard to discharge with groove M with cultivate with regard to the relation of groove L, can illustration discharging with the well width M2 of groove M be the length of 10 times～100 times of cultivating the well width L2 that uses groove L, particularly, can be more than illustration 10 μ m, preferred scope more than 30 μ m.
Like this, cultivate with groove L and discharge and can suitably design according to the size of the cell of cultivated expectation with groove depth (L1, M1) and the well width (L2, M2) of groove M.Enumerate concrete example, for example, known colibacillary size is roughly 0.5 μ m～1.0 μ m (width) * 1.5 μ m～7.0 μ m (length).When utilizing cell culture apparatus cultured continuously intestinal bacteria of the present invention, for example, for the cultivation of cell cultures substrate with for groove L, the scope that groove depth L1:1.0 μ m～1.5 μ m, well width L2:1.0 μ m～3.0 μ m can preference be shown, for discharging with for groove M, the scope of groove depth M1:5 μ m～20 μ m, well width M2:20 μ m～100 μ m can preference be shown.
The material of cell cultures substrate is such as exemplifying the resins such as the glass such as borosilicate glass, silica glass, polystyrene, plastics or silicon substrate etc.Wherein, the processibility of glass substrate, treatability excellence, thereby preferably.
In the situation that utilizing glass to make cell cultures substrate, a face with respect to sheet glass, for example by photolithography, will cultivate with groove L and discharge the pattern that forms photo-resist by the shape of groove M, recycle known method and carry out etching and form to cultivate with groove L and discharge and use groove M, thereby be made into cell cultures substrate.In addition, also can suitably to sheet glass, implement laser processing etc.In addition, as pattern, form, except photolithography, also can utilize electron beam directly to draw method etc.Cell cultures substrate can utilize surface treatment agent to process its surface, also can carry out the surface working of physical property and process.For example, can carry out cell cultures substrate surface or a surperficial part to give the processing of the functions such as wetting ability, lipophilicity, water-repellancy.For example, can give on surface silicon coating, functional group's coating, functional groups such as amino, isocyanate group, epoxy group(ing), carboxyl, hydroxyl, sulfydryl, silanol group.And, preferably utilize any modification of surfaces in vitamin H, avidin, Streptavidin etc.Or, also can utilize the cell contact matrix such as collagen, fibronectin, gelatin to apply the surface of cell cultures substrates.
Cultivate with groove L and discharge and be not limited to illustrative shape in Fig. 1 with the groove pattern of groove M.In cell cultures substrate, cultivate with groove L and be connected with the angle of discharging with groove M needs only with approximate right angle, for example also can between two discharges are with groove M, be formed with the form of the groove pattern of groove L for a cultivation for illustrated such the having of Fig. 2.In addition, as long as can suitably wash away the cell of cultivating with in groove L, discharging with groove M can not be also strictly with right angle, to connect with respect to cultivating with groove L, particularly, discharges with groove M and can allow the scope left and right at 90 ° ± 15 ° with respect to cultivating by the angle of groove L.In addition, cultivate with groove L and discharge and use groove M preferably linearly, but also can there is the crooked region of a part.In addition, in Fig. 1, cultivate with groove L and discharge, with the cross-sectional shape of groove M, form square, yet be not limited to this shape, for example, can be also the shape of the bottom bend of groove.
The semi-permeable membranes that cell culture apparatus of the present invention has is for covering groove and the discharge groove for cultivation of cell cultures substrate.As semi-permeable membranes, for example, can adopt the known semi-permeable membraness such as cellulose membrane.Semi-permeable membranes preferably utilizes any in vitamin H, avidin, Streptavidin to modify, and in this situation, cell cultures substrate also preferably utilizes any in vitamin H, avidin, Streptavidin to modify the surface of substrate.In the situation that cell cultures substrate utilizes biotin modification, the semi-permeable membranes that use utilizes avidin or Streptavidin to modify, in the situation that cell cultures substrate utilizes avidin, Streptavidin to modify, use utilize biotin modification semi-permeable membranes, thereby by the combination of vitamin H avidin, can be from groove L and the discharge groove M for cultivation of top encapsulated cell plate.Utilize this semi-permeable membranes, can from upper surface to cell, supply with all the time fresh nutrient solution.
In the situation that utilize semi-permeable membranes to cover the upper surface of cell cultures substrate, for example, method that can be below illustration.First, at the upper surface evaporation of cell cultures substrate or the silanol group of sputter and silicon or silicon oxide or chromium or aluminium or iron or titanium etc., carry out the film of silane coupled material.Then, on the surface of this film, such as there is importing amino or carboxyl or the sulfydryls such as bifunctional reagent of amino or carboxyl or sulfydryl at the other end by one end thering is silanol group, thereby make vitamin H form covalent linkage.Then, utilize avidin or Streptavidin to carry out the semi-permeable membraness such as film such as modified cellulose, this semi-permeable membranes contacted with film, by carry out vitamin H-avidin in conjunction with and upper surface that can encapsulated cell plate.It should be noted that, also can make surperficial avidin or the Streptavidin of the film of cell cultures substrate be combined with the vitamin H of semi-permeable membranes.It should be noted that, as long as semi-permeable membranes can be used groove and cultivate with groove from top with the discharge of good adhesion coating cell cultures substrate, may not be defined in the form of utilizing the combination of vitamin H-avidin.
Next, an embodiment of cell culture apparatus of the present invention is described.Fig. 3 (A) is the overall diagram exemplified with an embodiment of cell culture apparatus of the present invention, and a part is represented with section.Fig. 3 (B) is the sketch chart exemplified with the using method of semi-permeable membranes.In addition, the schematic diagram of the state of cell when Fig. 4 supplies with nutrient solution for the cell cultures substrate exemplified with to being covered by semi-permeable membranes, (A) is vertical view, is (B) sectional view.
As mentioned above, be formed with and cultivate with groove L and discharge and use groove M on cell cultures substrate 2, its upper surface is covered by semi-permeable membranes 3.As illustrated in Fig. 3 (B), semi-permeable membranes 3 at least covers the upper area of cultivating with groove L.By making to discharge with the both ends side of groove M in not covered by semi-permeable membranes 3 state of open, can be from discharging with one end supply nutrient solution of groove M and from the other end discharge nutrient solution.And, can keep cell in groove L in the cultivation being covered by semi-permeable membranes 3.
Cultivating with groove L and discharging with groove M around, on the surface of cell cultures substrate 2, be equipped with frame seal part S.Frame seal part has appropriate thickness, can preference be illustrated in the table back side and has the member of cementability, yet be not particularly limited.
The feed mechanism 4 of nutrient solution has liquid-feeding pump 41 (syringe), liquor charging liner 42, waste liquid tank 43.
Liquid-feeding pump 41 is connected with the communicating pores of one end of liquor charging liner 42 by liquid pushing tube, can send the required nutrient solution of cell and supplies with nutrient solutions to cell cultures substrate 2.
That is, liquor charging liner 42 has the function of the nutrient solution that remains fresh on cell cultures substrate 2.
In this embodiment, as shown in Fig. 3 (A), liquor charging liner 42 is adhered to the top of cell cultures substrate 2 across frame seal part S.Between cell cultures substrate 2 and liquor charging liner 42, be formed with space, when cultivating, this space is supplied with from liquid-feeding pump the nutrient solution coming and is full of.And the nutrient solution having passed through on cell cultures substrate 2 is sent to the waste liquid tank 43 being connected with this liquor charging liner 42 by the described communicating pores of liquor charging liner 42.Liquor charging liner 42 can illustrate the transparent pad for example being formed by PDMS (polydimethylsiloxane) by preference.The liquor charging liner 42 being formed by PDMS is for example by making and become case lid shape as follows: painting photoresist on silicon wafer, and utilize photolithography to make the casting mold of the convex that groove upset is formed, in this casting mold, inject PDMS resin and carry out hot briquetting.In addition, on liquor charging liner 42, such as de-bubble groove of removing the bubble in nutrient solution etc. also can be set at the bottom of case lid inner face.
And in Fig. 3 (A), the feed mechanism 4 of nutrient solution is configured to: the direction of the supply of nutrient solution and the discharge of cell cultures substrate 2 supplied with to cell cultures substrate 2 from liquid-feeding pump 41 are consistent with the length direction of groove M.
In addition, as shown in Figure 6, cell culture apparatus 1 of the present invention can with can observation of cell form cell cultures long-term observation device together with the microscopic examination mechanism 5 of cell on plate 2.As microscopic examination mechanism 5, such as enumerating the device that possesses the optical systems such as lens that the picture of the cell of the object of observation is amplified, particularly, can exemplify inverted microscope, opticmicroscope, fluorescent microscope, video record device, camera etc.And, also these microscopic examination mechanisms can be connected with PC etc. and carry out image processing.And, also can with for easily carrying out, together with the light irradiation device of cell observation, use.
Next, to having utilized the cell long-period culture method of cell culture apparatus of the present invention, an embodiment of cell cultures long-term observation method describes.
The liquid-feeding pump 41 of the feed mechanism 4 of Fig. 3, Fig. 5 is driven, and carry nutrient solution on cell cultures substrate 2.Because the direction of the liquor charging direction of the nutrient solution from liquid-feeding pump 41 to cell cultures substrate 2 and the discharge of cell cultures substrate 2 use groove M is consistent, therefore, from liquid-feeding pump 41, supply with the nutrient solution coming and flow into swimmingly with one end of groove M from discharging, be full of the internal space between liquor charging liner 42 and cell cultures substrate 2 and pass through on cell cultures substrate 2.For example, the liquor charging of nutrient solution can carry out with liquor charging speed in the scope of 0.5ml/hr～200ml/hr left and right.
Now, by utilizing semi-permeable membranes 3 to cover the upper surface of cell cultures substrates 2, thereby can be suppressed at while flowing nutritive medium on cell cultures substrate 2, in cultivating with groove L, keep cultured cells outflow and can supply with nutrient solution.Cell cultures substrate 2 is because the cultivation thin is connected with thick discharge groove M with the both sides of groove L, therefore exist such as in opposed two discharges by the slightly different situation of the flow velocity of the mobile nutrient solution of groove M etc.Therefore, in the situation that not using semi-permeable membranes 3, thereby may cause nutrient solution with flow velocity faster flow into cultivate with in groove L, cause cell from cultivate with in groove L to discharging the outflow with groove M.By utilizing semi-permeable membranes 3 to cover the cultivation groove L of cell cultures substrate 2, can suppress nutrient solution from discharging the inflow with groove L to cultivation with groove M, thereby can stably keep cultivating the cell with in groove L.
In addition, at the nutrient solution of the upper side flow of semi-permeable membranes 3 by seeing through via semi-permeable membranes 3, diffusion phase supplies with the cell in groove L for remaining on to cultivate.Therefore, for example, cultivation is become to such an extent that grow to 30 μ m above in the situation that with groove is L shaped, also can cultivate with maintaining stable and uniform envrionment conditions in groove L.And, by making culture environment even, can check more accurately the response of cell to medicine etc.
The schematic diagram of the state of cell when Fig. 4 supplies with nutrient solution for the cell cultures substrate exemplified with to being covered by semi-permeable membranes.It should be noted that, in Fig. 4, omitted and cultivated by groove L and the diagram of discharging the boundary line of the connection section of using groove M.Fig. 4 (A) is vertical view, and Fig. 4 (B) is sectional view.
By the supply of the nutrient solution that carries out via semi-permeable membranes 3, can stably cultivate with the cell in groove L, make it be geometricprogression and breed.And, when becoming cells fill and cultivate with state in groove L, utilization is mobile nutrient solution in cultivation is used groove M with the both sides of groove L with the discharge of the angle connection of approximate right angle, a part that is positioned near the cell in the two ends (discharge with groove M and cultivate the connection section with groove L) of cultivating with groove L is washed away with groove M to discharge, and discharge together with nutrient solution and to waste liquid tank 43 recovery.Therefore, can the quantity that maintain regulation by the quantity of the cell in groove L will be cultivated, thereby can solve the nutrient consumption of the cell in the past producing along with the process of time, problems such as the savings of refuse, pericellular environmental changes, the cell cultures long-term observation device of microscopic examination mechanism 5 has been used in utilization, can go through 200 continuous cultivation, the observations of above cell from generation to generation.Thus, can the long-standing time series data based on do not have in the past measure the speed of growth of a clone, the size of the fluctuation of gene expression dose, the information of autocorrelative function.And, owing to going through for 100 above times of generation, be equivalent to occur the time ruler along with the Evolving State of genotypic variation, the Study on Evolution that therefore cell culture apparatus of the present invention, cell cultures long-term observation device can be applied to be difficult to up to now carry out experimental confirmation, check.
And, form the cell cultures substrate of cell culture apparatus of the present invention because the two ends cultivating with groove L are connected with discharge groove M, therefore cultivate and there is mobility with the cell in groove L, thereby can avoid parent cell as " Mother machine " in the past to residue in the phenomenon of cultivating with groove L.Therefore, can suppress to follow the variation of cultivating the physiological status producing with the cell senescence in groove L.
And, because cell residual in cultivating with groove L is for cultivating from being present in the cell splitting off with the cell in groove L (parent cell), therefore by the image analysis that carries out being undertaken by microscopic examination mechanism 5, computer etc., can cell cultures long-term observation device follow the trail of the history of specific cell and observe, resolve (pedigree).
In addition, cell culture apparatus of the present invention, cell cultures long-term observation device also can be for the detections of transgenation.Particularly, while being designed to, at certain specific position, the transgenations such as phase shift mutation, point mutation have occurred by use, the fluorescence protein that is disposed at this position cell strain of expressing with correct arrangement, can in the mode that cell starts to send fluorescence by the expression of fluorescence protein, cell be detected and not be killed the situation of suddenling change has occurred being present in to cultivate while there is this sudden change with the cell in groove.By this cell of Continuous Observation between long-term in cell culture apparatus, can obtain the Frequency of this transgenation, the information such as time of origin in mitotic cycle.
Fig. 5 is exemplified with the overall diagram of another embodiment of cell culture apparatus of the present invention, and a part is represented with section.Omission is to the explanation with the common part of the mode shown in Fig. 3, Fig. 4.
In the illustrated cell culture apparatus 1 of Fig. 5, at liquor charging liner 42, be connected with a plurality of liquid-feeding pumps 41, can to the cell cultures substrate 2 being covered by semi-permeable membranes 3, supply with nutrient solution from each liquid-feeding pump.In this embodiment, for example, in each liquid-feeding pump 41, put into the nutrient solution of heterogeneity, appropriate time in cultured continuously switches the liquid-feeding pump using, thereby can with the cell in groove L, apply the variation of various envrionment conditionss to remaining on the cultivation of cell cultures substrate 2, can check being for a long time applied with the impact of cell of the variation of envrionment conditions.
Fig. 6 is exemplified with the overall diagram of an embodiment of cell cultures long-term observation device of the present invention, and a part is represented with section.Omission is to the explanation with the common part of the embodiment shown in Fig. 3, Fig. 5.
The illustrated cell cultures long-term observation of Fig. 6 device 1a possesses cell culture apparatus 1 and microscopic examination mechanism 5.
In this embodiment, as being suitable for the speed of growth of cell as above, the expression of fluorescence protein etc. through time the microscopic examination mechanism 5 observed, use the handstand type electric microscope that possesses fluorescent microscope function.
Handstand type electric microscope (microscopic examination mechanism 5) possesses: light source 51a is observed in bright field, light source 51b, automatic shutter 52a, automatic shutter 52b, condensing lens 53, dichroscope 54, object lens 55 and XY platform 56 for Fluirescence observation.XY platform 56 has peristome A, and the cell cultures substrate 2 of cell culture apparatus 1 is positioned at peristome A with groove L mode to maintain the cultivation of cell is positioned on XY platform 56.
Be held in cultivate with the cell utilization of groove L by electric motor driven XY platform 56 can automated movement to position that wish is observed.Below the peristome A of XY platform 56, be adjacent to dispose object lens 55.By make XY platform 56 respectively along X, the Y direction of crosscut optical axis move and make object lens 55 along Z-direction up and down direction move, can adjust the relative position of XY platform 56 and object lens 55.And, by making XY platform 56 not only move also independent movement in Z-direction in X, Y direction, also can regulate the relative distance of XY platform 56 and object lens 55.
Handstand type electric microscope possesses bright field transmission illumination system, reflective illumination system, camera system in the inside of not shown main body cover.Bright field transmission illumination system configuration is in the position of the top side of height location than XY platform 56, and reflective illumination system and camera system are configured in than the height location of XY platform 56 position on the lower.
Bright field transmission illumination system is for utilizing the observation that sees through light.Bright field transmission illumination system comprises bright field and observes light source 51a, automatic shutter 52a, condensing lens 53.Light source 51a is observed in bright field can use halogen lamp etc.The state that the light penetrating from this bright field observation light source 51a is opened at automatic shutter 52a, by condensing lens 53, towards vertical direction below, to being held in the cultivation of the cell cultures substrate 2 of mounting on XY platform 56, with the cell in groove L, irradiate.
Reflective illumination system is for utilizing the observation of fluorescence.Reflective illumination system comprises light source 51b, automatic shutter 52b, dichroscope 54 for Fluirescence observation.Fluirescence observation can be used mercury lamp etc. with light source 51b.Reflective illumination system can also possess in addition heat absorption filter, condensing lens, for making, become the optical systems such as exciter filter of the exciting light of specific short-wave band from Fluirescence observation with the light of light source 51b.The state of opening at automatic shutter 52b with the light that light source 51b penetrates from this Fluirescence observation, by dichroscope 54 and object lens 55, towards vertical direction top, from peristome A, to being held in the cultivation of the cell cultures substrate 2 loading at XY platform 56, with the cell in groove L, irradiate.
Camera system comprises the camera 57 of installing towards dichroscope 54.Camera 57 can use for example CCD camera.
In addition the electric microscope of handstand type, is equipped with power subsystem, motor drive circuit plate etc.Power subsystem comprise bright field observe the power supply of light source 51a, for control the systems such as electric motor that pack handstand type electric microscope into power supply, Fluirescence observation with the power supply of light source 51b etc.Motor drive circuit plate such as to for driving the electric motor of XY platform 56, for driving the electric motor of the power zoom mechanism of camera system, for driving the Fluirescence observations such as mercury lamp to control with electric motor of the aperture of light source 51b etc. in X, Y direction.
User can and utilize keyboard, mouse to carry out various operations, setting to handstand type electric microscope by the computers such as PC 58.
Can optionally use transmission illumination system and reflective illumination system.When selecting transmission illumination system, the picture of using the cell in groove L is cultivated in being maintained at of obtaining by the transmission illumination light from bright field observation light source 51a, from peristome A, by object lens 55, by dichroscope 54, reflected, the camera 57 being configured towards horizontal direction is taken in.
On the other hand, when selecting reflective illumination system, by the picture based on fluorescence of cultivating with the cell in groove L that is maintained at obtaining with the irradiation of the exciting light of light source 51b from Fluirescence observation, from peristome A, by object lens 55, by dichroscope 54, reflected, be imported into towards the camera 57 of horizontal direction configuration and take in.
In addition,, although not shown, handstand type electric microscope possesses for the eyepiece with visual inspection cell with by the optical system of eyepiece and object lens 55 optical bond.This optical system comprises speculum, relay lens, light path switching prism.For example, when utilizing camera 57 to observe, light path is switched prism and is inserted on observation optical axis, and object lens preimage is switched prismatic reflection by light path, can utilize camera 57 to observe.On the other hand, when observing by naked eyes, light path is switched prism and is exited from observation optical axis, and object lens preimage is reflected mirror and reflects towards eyepiece.In addition, object lens preimage is changed by relay lens, thereby can with naked eyes, observe by eyepiece.
Utilize the as above cell culture apparatus 1 of structure, can carry out following cell observation.
Same with the embodiment of Fig. 3, Fig. 5, utilize the feed mechanism 4 of nutrient solution, to being adhered to across frame seal part S between the liquor charging liner 42 of top of cell cultures substrate 2 and cell cultures substrate 2, supply with nutrient solution.It should be noted that, in this embodiment, at liquor charging liner 42, be provided with the groove 44 for de-bubble that the groove of the bubble in nutrient solution is removed in above-mentioned conduct.
In this embodiment, use handstand type electric microscope as microscopic examination mechanism 5, it is very important therefore making the observation place of microscopic examination mechanism 5 be positioned under cell culture apparatus 1.
Thus, the bubble being supplied in the nutrient solution between liquor charging liner 42 and cell cultures substrate 2 is removed with groove 44 upward by the de-bubble of cell cultures substrate 2 tops, from the below of the cell cultures substrate 2 with its opposition side, utilize object lens 55 can access by the peristome A of XY platform 56 intensified image that is maintained at the cell in cultivation use groove L.
Therefore, can prevent from hindering at the bubble of cell culture apparatus 1 interior generation the situation of utilizing the observation that microscopic examination mechanism 5 carries out, thereby also can carry out long-term successive cell observation, for example 200 long-term successive cells are from generation to generation observed.
By the supply of the nutrient solution that carries out via semi-permeable membranes 3, can stably cultivate with the cell in groove L, make it be geometricprogression and breed.And, when becoming cells fill and cultivate with state in groove L, utilization washes away with groove M to discharge by a near part for the cell two ends of groove L being positioned at cultivation with nutrient solution mobile in groove M in the discharge of cultivating with the both sides of groove L connect with the angle of approximate right angle, and discharges together with nutrient solution.
Like this, by cultured cells in cultivating with groove L is irradiated to any in transmission illumination system and reflective illumination system, can observe the picture that uses object lens 55 to amplify by camera 57 or eyepiece.
In the observation that utilizes transmission illumination system to carry out, particularly, can observe the size of cell as above, particularly the variation of the cell size in a clone, the speed of growth etc.
In the Fluirescence observation that utilizes reflective illumination system to carry out, particularly, can observe expression level and the variation thereof of the fluorescence protein at cell interior as above, the fluoroscopic image of the cell that dyeed and variation thereof etc.
; because cell residual in cultivating with groove L is for cultivating from being present in the cell splitting off with the cell in groove L (parent cell); therefore by utilizing the parsing etc. of microscopic examination mechanism 5, computer 58, can follow the trail of observing historically, resolving of specific cell.
For example, by utilize camera 57 through time obtain fluoroscopic image of fluorescence protein etc., and be recorded in computer 58, can carry out Time delay measurement.By utilizing special-purpose image analysis software in computer 58, this time delay image to be resolved, can obtain to obtain image in the size of the cell that contains, average etc. the relevant time sequence information of inner fluorescent intensity.Thus, can the long-standing time series data based on do not have in the past measure the speed of growth of a clone, the information of the size of the fluctuation of gene expression dose, autocorrelative function etc.
The present invention is not limited to above embodiment.It is self-evident can taking various forms to its detail section.
[embodiment]
Below, by embodiment, illustrate in greater detail the present invention, yet the present invention is not limited to these embodiment.
Making of < embodiment 1> cell cultures substrate
As for the colibacillary cell cultures substrate of cultured continuously, at glass substrate (60mm (length) * 24mm (width) * 0.17mm (thickness)), above with reticulation, arrange and cultivate (groove depth L1:1.0 μ m, well width L2:3.0 μ m, length 30 μ m with groove L, article 50), discharge with groove M (M1: groove depth 17 μ m, M2: well width 60 μ m, length 5000 μ m, 20) (with reference to Fig. 1).
The covering that < embodiment 2> is undertaken by semi-permeable membranes
Utilize the surface of the cell cultures substrate that biotin modification makes in embodiment 1.On the other hand, as semi-permeable membranes, use the Mierocrystalline cellulose semi-permeable membranes processed of width 1mm * length 1mm, and utilize Streptavidin modification of surfaces.And, at the groove L for surperficial cultivation from cell cultures substrate, above discharging with groove M, drip the nutrient solution that comprises colibacillary 1 μ l, the cultivation that utilizes semi-permeable membranes encapsulated cell plate is with groove L and a part of region of middle body of discharging the top of use groove M.
Cultivation, the observation of < embodiment 3> cell
Utilize cell culture apparatus of the present invention, cell is cultivated continuously, observed.Particularly, using (バ イオラツ De society (Bio-Rad) system: SLF-0201) be sealed on cell cultures substrate by the mode of groove to surround to cultivate with groove and discharge, then paste the liquor charging liner of PDMS system at frame seal part upper surface of the frame seal part that is four frame shapes as two sides band.One of being installed in two silicon tubulations of liquor charging liner of PDMS system is linked with syringe (liquid-feeding pump), another root is imported in waste liquid tank (with reference to Fig. 3).By syringe, the intestinal bacteria of the flow velocity glucose that continuous flow action contains 0.2% weight ratio as nutrition source continuously of 2ml/h of take are cultivated and use M9 minimal medium.By this cell culture apparatus being configured on the platform of handstand type electric microscope and the cell cultures long-term observation device shown in pie graph 6, and utilize 100 times of phase contrast objectives of multiplying power to observe with interval time delay in 2 minutes the cell of cultivating with in groove.
The intestinal bacteria of using in observation are particularly W3110 bacterial strain, and have the plasmid of the mode express fluorescent protein matter (GFP) to be controlled by the promotor of rpsL gene.
In Time delay measurement, obtain the fluoroscopic image of the GFP of cell, and be recorded on PC.By utilizing image analysis software I mageJ (http://rsbweb.nih.gov/ij/) to resolve this time delay image, and obtain to obtain image in the size of the cell that contains, average (IC that is equivalent to GFP) of inner fluorescent intensity relevant time sequence information.
Fig. 7 records a part for the consecutive image of the situation of the cell in groove for the cultivation that is present on cell cultures substrate.
As shown in Figure 7, can confirm, while carrying out cultured continuously when cell cultures substrate is supplied with to nutrient solution, be present in cultivate with in the cell in groove, be present in a part of cultivating with the cell at the two ends of groove and moderately to discharge, with groove, washed away and disappear from picture.And, can also confirm, utilize the groove shape of cell cultures substrate and semi-permeable membranes can by cytotostatic remain on and cultivate with cultivating in groove L.In addition, by the supply of the cytotropic nutrient solution that carries out via semi-permeable membranes, can cultivate with maintaining in groove L under stable and uniform environment.
Can confirm, by moderately washing away with groove to discharging with the cell at the two ends of groove being present in cultivation, can the quantity that maintain regulation by the quantity of the cell in groove L will be cultivated, thereby can solve the nutrient consumption of the cell in the past producing along with the process of time, the problems such as variation of the savings of refuse, pericellular environment can be carried out continuous cultivation, the observation of cell.And, can also confirm, can avoid parent cell to residue in cultivation and use groove L, thereby can not occur along with variation aging and physiological status that produce, and long-term cultivation is cultivated with the cell in groove L.
Fig. 8 is for drawing the variation of 55 cell sizes from generation to generation have a clone obtaining by image analysis and at the figure of the variation of the GFP of cell interior expression level (IC).
As shown in Figure 8, can confirm, utilize cell culture apparatus of the present invention, can carry out the cultured continuously of long-term cell.Thus, can confirm, can measure the speed of growth (variation of cell size) of a clone, the information such as size of the fluctuation of gene expression dose by the long-standing time series data based on do not have in the past.
The frequency of < embodiment 4> protein expression level distributes
The cell culture apparatus of the liquor charging liner with cell cultures substrate, PDMS system similarly to Example 3, syringe (liquid-feeding pump) and waste liquid tank is configured on the platform of handstand type electric microscope, to cultivating, with the intestinal bacteria of the express fluorescent protein matter GFP in groove, carries out time delay observation.By syringe, the intestinal bacteria of the flow velocity glucose that continuous flow action contains 0.2% weight ratio as nutrition source continuously of 2ml/h of take are cultivated and use M9 minimal medium.Culture temperature during by observation maintains 37 ℃, and observes cultivating with the time delay that the cell in groove carries out 5000 minutes with 1 minute interval.
GFP mean fluorecence briliancy in the cell of all cells in all moment that mensuration is observed, thus the frequency that determines the GFP expression level of inferring out accordingly distributes.Fig. 9 represents its result.
The frequency of < embodiment 5> cell size distributes
In the time delay of embodiment 4 is observed, the frequency of having measured cell size distributes.Its result as shown in figure 10.
The frequency of < embodiment 6> growth rate distributes
In the time delay of embodiment 4 is observed, the frequency of having measured growth rate distributes.For the growth rate of a cell, with exponential function C * 2
ktexpression is from splitting into the variation of the cell size of division end, the growth rate using its index k as each cell.Wherein, C represents constant, and t represents the time.Its result as shown in figure 11.
< embodiment 7> distributes generation time
In the time delay of embodiment 4 is observed, the frequency of having measured generation time distributes.Generation time is for each cell is from splitting into the needed time till next time division.Its result as shown in figure 12.
The autocorrelative function of < embodiment 8> protein expression level
In the time delay of embodiment 4 is observed, measured the autocorrelative function of protein expression level.By when the protein expression level of t is made as x (t) sometime, by calculating following formula, obtained and the autocorrelative function A (t) that has passed through the moment of time t.
[changing 1]
Wherein, E[], V[] represent respectively mean value and discrete.Its result as shown in figure 13.This figure has drawn the figure of A (τ) with respect to τ.
The distribution in < embodiment 9> cell fission age
In the time delay of embodiment 4 is observed, the frequency of having measured cell fission age distributes.Cell fission represents from being about to split into the current time age.Its result as shown in figure 14.
The cell lineage that < embodiment 10> observes in groove an observation
In the time delay of embodiment 4 is observed, measured the cell lineage of observing in groove an observation.Its result as shown in figure 15.The branching representation cell fission of series, the breaking part of series represents that cell is flushed away.
< embodiment 11>
The cell culture apparatus of the liquor charging liner with cell cultures substrate, PDMS system similarly to Example 3, syringe (liquid-feeding pump) and waste liquid tank is configured on the platform of handstand type electric microscope, to cultivating, with the cell in groove, carries out time delay observation.The intestinal bacteria that replace embodiment 3 with the embryonic stem cell of mouse, by syringe pump, with the flow velocity of 2ml/h, replace intestinal bacteria and cultivate with M9 minimal medium that continuing continuously flows not break up to maintain uses serum free medium ESF7.Bonding in order to promote with the embryonic stem cell of mouse, to described glass baseplate surface coating collagen or E cadherin.By handstand type electric microscope and utilize 20 times of phase contrast objectives of multiplying power, with 15 minutes intervals, cultivate and carry out time delay observation with the embryonic stem cell of the mouse in groove.
Its result is, can confirm, while carrying out cultured continuously when cell cultures substrate is supplied with to nutrient solution, be present in cultivate with in the embryonic stem cell of the mouse in groove, be present in a part of cultivating with the embryonic stem cell of the mouse at the two ends of groove and with groove, washed away to discharge by appropriateness.
And, can also confirm, utilize groove shape and the semi-permeable membranes of cell cultures substrate, the embryonic stem cell of mouse stably can be remained on and cultivate with cultivating in groove L.
Can confirm, the supply of the nutrient solution by the embryonic stem cell to mouse that carries out via semi-permeable membranes, can will cultivate with maintaining in groove L under stable and uniform environment, by moderately washing away with groove to discharging with the embryonic stem cell of the mouse at the two ends of groove being present in cultivation, cultivation can be maintained to the quantity of regulation by the quantity of the embryonic stem cell of the mouse in groove L, and then can carry out continuous cultivation, the observation of cell.And, can also confirm, can avoid parent cell to residue in cultivating use groove L, thereby can not occur that long-term cultivation is cultivated the embryonic stem cell of the mouse in use groove L along with the variation of the physiological status of aging generation.
< comparative example 1>
Can surface at glass substrate is formed to cultivation that groove depth L1 is less than the size of cell carry out colibacillary cultivation with groove L and be studied.Particularly, with reticulation, arrange and cultivate with groove L (L1: groove depth 0.5 μ m, L2: well width 2.0 μ m, length 30 μ m, 50), discharge groove M (M1: groove depth 17 μ m, M2: well width 60 μ m, length 5000 μ m, 20).
And, utilize similarly to Example 2 semi-permeable membranes to cover cell cultures substrate, with apparatus structure similarly to Example 3, cell cultures substrate is supplied with to nutrient solution, cell is observed.
Result, as shown in Figure 16 (A), is cultivated and by semi-permeable membranes, is crushed and be out of shape with the cell C in groove L, can not carry out the observation under standard state.
< comparative example 2>
Can over the big or small cultivation of 2 times of cell size (thickness), with groove L, carry out colibacillary cultivation be studied the surface formation groove depth L1 at glass substrate.Particularly, with reticulation, arrange and cultivate with groove L (L1: groove depth 2.0 μ m, L2: well width 2.0 μ m, length 30 μ m, 50), discharge groove M (M1: groove depth 17 μ m, M2: well width 60 μ m, length 5000 μ m, 20).
And, utilize similarly to Example 2 semi-permeable membranes to cover cell cultures substrate, with apparatus structure similarly to Example 3, cell cultures substrate is supplied with to nutrient solution, cell is observed.
Result, as shown in Figure 16 (B), can be confirmed, cultivates with the cell C in groove L and is not stably kept and with groove L, to discharging, use groove M flow process from cultivating, and causes being difficult to carrying out cultured continuously.
[nomenclature]
1 cell culture apparatus
1a cell cultures long-term observation device
2 cell cultures substrates
3 semi-permeable membraness
4 feed mechanisms
5 microscopic examination mechanisms
41 liquid-feeding pumps
42 liquor charging liners
43 waste liquid tanks
44 de-bubble grooves
Light source is observed in 51a bright field
51b Fluirescence observation light source
52a automatic shutter
52b automatic shutter
53 condensing lenses
54 dichroscopes
55 object lens
56 XY platforms
57 cameras
58 computers
L cultivation groove
M discharge groove
A peristome
S frame seal part
Claims (9)
1. a cell culture apparatus, it has the feed mechanism of cell cultures substrate, semi-permeable membranes, nutrient solution, and described cell culture apparatus is characterised in that,
Cell cultures substrate has thin cultivation for keeping culturing cell with groove with for by the thick discharge groove that keeps cultured cells to discharge in groove in this cultivation on surface, and, described cultivation is connected with groove with discharge with the two ends of groove, discharge is cultivated and ratio cultivation groove depth thick with groove with groove ratio
Semi-permeable membranes is used for covering groove and the discharge groove for cultivation of cell cultures substrate,
The feed mechanism of nutrient solution can be supplied with nutrient solution continuously to the cell cultures substrate being covered by semi-permeable membranes.
2. cell culture apparatus as claimed in claim 1, is characterized in that,
Described semi-permeable membranes can be by vitamin H avidin in conjunction with covering cell cultures substrate.
3. cell culture apparatus as claimed in claim 1, is characterized in that,
The feed mechanism of described nutrient solution has liquor charging liner.
4. a cell cultures long-term observation device, is characterized in that,
This cell cultures long-term observation device possess the cell culture apparatus described in any one in claims 1 to 3 and can observation of cell plate on the microscopic examination mechanism of cell.
5. cell cultures long-term observation device as claimed in claim 4, is characterized in that,
Microscopic examination mechanism is inverted microscope.
6. a cell long-period culture method, is characterized in that,
Utilize the cell culture apparatus described in claim 1.
7. cell long-period culture method as claimed in claim 6, wherein, this cell long-period culture method comprises:
The cell of expectation is remained on to the operation in groove for cultivation of cell cultures substrate;
The operation of groove is used in the cultivation that utilizes semi-permeable membranes to cover cell cultures substrate with groove and discharge;
Utilize feed mechanism to carry continuously nutrient solution to cell cultures substrate, via semi-permeable membranes, to the cultivation that is maintained at cell cultures substrate, with the cell in groove, supply with nutrient solution, and, utilize the discharge that is connected at the two ends with cultivating with groove with nutrient solution mobile in groove will cultivate a part with the cell in groove to discharge the operation with groove discharge.
8. a cell cultures long-term observation method, is characterized in that,
This cell cultures long-term observation method is utilized the cell cultures long-term observation device described in claim 4.
9. cell cultures long-term observation method as claimed in claim 8, wherein, this cell cultures long-term observation method comprises:
The cell of expectation is remained on to the operation in groove for cultivation of cell cultures substrate;
The operation of groove is used in the cultivation that utilizes semi-permeable membranes to cover cell cultures substrate with groove and discharge;
Utilize feed mechanism to carry continuously nutrient solution to cell cultures substrate, via semi-permeable membranes, to the cultivation that is maintained at cell cultures substrate, with the cell in groove, supply with nutrient solution, and, utilize the discharge that is connected at the two ends with cultivating with groove with nutrient solution mobile in groove will cultivate a part with the cell in groove to discharge the operation with groove discharge;
Utilize the operation of the cell on microscopic examination mechanism observation of cell plate.
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| JP2011156767 | 2011-07-15 | ||
| JP2011-156767 | 2011-07-15 | ||
| PCT/JP2012/068005 WO2013011962A1 (en) | 2011-07-15 | 2012-07-13 | Cell culture device, device for long-term monitoring of cell culture, method for long-term cell culture, and method for long-term monitoring of cell culture |
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| EP (1) | EP2733199B1 (en) |
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| KR (1) | KR101391679B1 (en) |
| CN (1) | CN103649302B (en) |
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| WO (1) | WO2013011962A1 (en) |
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Also Published As
| Publication number | Publication date |
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| EP2733199A1 (en) | 2014-05-21 |
| EP2733199A4 (en) | 2015-04-15 |
| CA2842023C (en) | 2017-02-28 |
| CN103649302B (en) | 2015-04-15 |
| US9139807B2 (en) | 2015-09-22 |
| WO2013011962A1 (en) | 2013-01-24 |
| KR101391679B1 (en) | 2014-05-07 |
| EP2733199B1 (en) | 2016-06-29 |
| CA2842023A1 (en) | 2013-01-24 |
| JP5231684B1 (en) | 2013-07-10 |
| KR20140019475A (en) | 2014-02-14 |
| US20140147879A1 (en) | 2014-05-29 |
| JPWO2013011962A1 (en) | 2015-02-23 |
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